Isomaltooligosaccharides to inhibit avian pathogenic intestinal bacteria

ABSTRACT

Isomaltooligosaccharides (IMOs) produced by  Leuconostoc mesenteroides  ATCC 13146 fermentation with a sucrose:maltose ratio of 2:1 have been discovered to be effective prebiotics in mixed cultures of microbial populations, including cultures from chicken ceca. Surprisingly in mixed microbial cultures this IMO composition proved as effective as FOS. Thus, these IMOs can be used as effective prebiotics for both birds and mammals. Moreover, the IMOs were discovered to be effective non-competitive inhibitors of α-glucosidase. These IMOs also will be useful, as an α-glucosidase inhibitor, in a therapeutic application for several diseases, including obesity, diabetes mellitus, pre-diabetes, gastritis, gastric ulcer, duodenal ulcer, caries, cancer, viral disease such as hepatitis B and C, HIV, and AIDS. A diet with 5-20% IMOs was also shown to reduce the abdominal fat tissue in mammals.

This is a divisional of co-pending application Ser. No. 10/848,981,filed May 19, 2004, which claims the benefit of U.S. ProvisionalApplication No. 60/471,942 filed May 20, 2003.

This invention pertains to the use of maltosyl-isomaltooligosaccharidesas a dietary supplement for birds and mammals, specifically, to promotethe growth of beneficial intestinal microbes, inhibit the growth ofpathogenic intestinal microbes, and for therapeutic intervention indiseases such as diabetes by inhibiting the activity of α-glucosidase toslow the rate of glucose release from carbohydrates and thereby reducethe uptake of glucose.

Prebiotics are nondigestible food ingredients that selectively stimulatethe growth and/or activity of beneficial microbial strains (probiotics)residing in the host intestine. See R. Barrangou et al., “Functional andcomparative genomic analyses of an operon involved infructooligosaccharide utilization by Lactobacillus acidophilus,” Proc.Natl. Acad. Sci. USA, vol. 100, pp. 8957-8962 (2003). It is believed theability of these probiotics to catabolize oligosaccharides (two to tenmonosaccharide units linked with glycosidic bonds) is a key factor inbestowing beneficial health effects. Certain oligosaccharides are usedas prebiotics. They are resistant to metabolism and adsorption in thesmall intestine and ultimately positively influence the composition ofmicroflora in the large intestine. Oligosaccharides are also widely usedin foods such as soft drinks, cookies, cereals, candies, and dairyproducts. Other applications for oligosaccharides such as ananti-cariogenic agent or a low sweetness humectant have been explored.See S. K. Yoo, “The production of glucooligosaccharides by Leuconostocmesenteroides ATCC 13146 and Lipomyces starkeyi ATCC 74054, Ph.D.Dissertation, Louisiana State University (1997).

Oligosaccharides used as prebiotics are currently produced either byextraction from plant sources, acid or enzymatic hydrolysis ofpolysaccharides or enzymatic synthesis by transglycosylation reactions.See P. Monsan et al., “Oligosaccharide feed additives,” In: R. J.Wallace and A. Chesson (eds) Biotechnology in animal feeds and animalfeeding, pp. 233-245, VCH Velagsgesellshaft mbH, Weinheim, Germany(1995).

Types of Oligosaccharides

Types of oligosaccharides include fructooligosaccharides (FOS),glucooligosaccharides (GOS), and α-galactooligosaccharides. Thedifferences in structures are illustrated in FIG. 1.Fructooligosaccharides (FOS) have attracted serious commercial interestas prebiotics. They are composed of a D-glucopyranose unit at thenon-reducing end (G) linked via an α-1,2 linkage to two or moreβ-2,1-linked fructosyl units (F). This group includes 1-kestose (GF2),nystose (GF3), and 1F-fructofuranosyl nystose (GF4). Many of theoligosaccharides marketed commercially are FOS, e.g., Raftilose andNutraflora in the United States.

α-Galactooligosaccharides, which are α-galactosyl derivatives ofsucrose, are present in many legume seeds. Mono-, di-, andtri-α-galactosylsucrose, known respectively as raffinose, stachyose, andverbascose, are produced by extraction from plants, particularlysoybeans. These oligosaccharides are known to be, in part, responsiblefor the flatulence and diarrhea that follows consumption of beans,because of the absence of an α-galactosidase in the gastrointestinaltracts of humans and animals.

Glucooligosaccharides (GOS) is a generic term for poly-glucoseoligomers. GOS may contain a number of different linkages and aregenerally obtained from starch hydrolysates (maltose and maltodextrins)through the action of the α-transglucosidase (EC 2.4.1.24) fromAspergillus sp. Glucooligosaccharides can also be produced byrestricting polymer size during the fermentation process. A subcategoryof GOS is the α-isomaltooligosaccharides (IMO) which contain α-1,6 bondsin their main chain. See H. J. Koepsell et al., “Enzymatic synthesis ofdextran. acceptor specificity and chain initiation,” J. Biol. Chem.,vol. 200, pp. 793-801 (1952). Dextransucrase (EC 2.4.1.5), an enzymeproduced mainly by species of Leuconostoc and Streptococcus, catalyzesthe synthesis of high molecular weight glucans (dextrans).

Oligosaccharides as Prebiotics

Ingested oligosaccharides (prebiotics) are capable of reaching the colonwithout being digested. It has been proposed that fructooligosaccharidesare preferentially utilized by Lactobacilli and Bifidobacterial specieswhich are considered beneficial species of the human intestinal tract.See H. Kaplan et al., “Fermentation of fructooligosaccharides by lacticacid bacteria and Bifidobacteria,” Appl. Environ. Microbiol., vol. 66,pp. 2682-84 (2000). Substituting fructooligosaccharides as a carbonsource would preferentially increase the concentration of Lactobacillusand Bifidobacteria species with a concomitant rise in the intestinalproduction of lactic acid and short-chain fatty acids (SCFA). Both theseproducts would have the net effect of lowering the pH in the largeintestine. This appears to be one mode by which beneficial species canout-complete and indeed help prevent the establishment of undesirablepathogenic organisms such as Salmonella. See B. J. Juven et al.,“Antagonistic effects of Lactobacilli and Pediococci to controlintestinal colonization by human enteropathogens in live poultry,” J.Appl. Bacteriol., vol. 70, pp. 95-103 (1991). The fructooligosaccharidesmay also interact with carbohydrate receptors present on the surface ofeither microbial or epithelial cells, affecting cell adhesion andimmunomodulation. See P. J. Naughton et al., “Effects of nondigestibleoligosaccharides on Salmonella enterica Serovar Typhimurium andnonpathogenic Escherichia coli in the pig small intestine in vitro,”Appl. Environ. Microbiol., vol. 67, pp. 3391-95 (2001).

Fructooligosaccharides, galactooligosaccharides, and soybeanoligosaccharides were found not to be digested by enzymes secreted bysmall intestine, but to be fermented by certain microorganisms found inhuman and livestock intestines, especially by the Bifidobacterium sp.See. H. Tomomatsu, “Health effects of oligosaccharides,” Food Technol.,vol. 48, pp. 61-65 (1994). There are numerous reports regarding thestimulating effects of fructooligosaccharides on the growth of probioticstrains. See P. Monsan et al., 1995; and M. Gmeiner et al., “Influenceof a symbiotic mixture consisting of Lactobacillus acidophilus 72-4 anda fructooligosaccharide preparation on the microbial ecology sustainedin a simulation of the human intestinal microbial ecosystem (SHIMEreactor),” Appl. Microbiol. Biot., vol. 53, pp. 219-223 (2000). DietaryFOS have been reported to be effective in reducing the numbers of theharmful bacteria, E. coli, in the intestine of piglets, but did notreduce numbers of Salmonella. See P. J. Naughton et al., “Effects ofnondigestible oligosaccharides on Salmonella enterica SerovarTyphimurium and nonpathogenic Escherichia coli in the pig smallintestine in vitro,” Appl. Environ. Microbiol., vol. 67, pp. 3391-95(2001). However, in the same study, commercially availableglucooligosaccharides (GOS), another oligosaccharide, showed no effecton either genus of bacteria.

In studies of in vitro fermentation characteristics using human fecalmaterial, small intestinal digestibility, and effects on fecal microbialpopulations in dogs, GOS (containing α-1,2, α-1,4 and α-1,6 linkages)and FOS produced short chain fatty acids in human fecal material morerapidly than other substrates, such as gum arabic, guar gum and guarhydrolysate. GOS also appeared to be indigestible in the smallintestine, while supplying a carbon source for bacterial fermentationsin the large intestine of cannulated dogs. See E. A. Flickinger et al.,“Glucose-based oligosaccharides exhibit different in vitro fermentationpatterns and affect in vivo apparent nutrient digestibility andmicrobial populations in dogs,” J. Nutr., vol. 130, pp. 1267-1273(2000). When the viable count of Bifidobacterium infantis and B. longum,and changes in pH due to various carbohydrate-supplemented soymilks weremonitored, B. longum showed a significantly (P<0.05) higher count on acrude isomaltooligosaccharide (75%) supplemented soymilk than in thecontrol (soymilk without the added supplement) at the end offermentation. See C-C. Chou et al., “Growth of Bifidobacteria in soymilkand their survival in the fermented soymilk drink during storage,” Int.J. Food Microbiol., vol. 56, pp. 113-121 (2000). Another study showedthat GOS was only 20% digested by germfree rats. See P. Valette et al.,“Bioavailability of new synthesized glucooligosaccharides in theintestinal tract of gnotobiotic rats,” J. Sci. Food Agric., vol. 62, pp.121-127 (1993). Dietary isomaltooligosaccharides (13.5 g/day for 14days) were reported to increase fecal Bifidobacteria levels (P<0.05) inhealthy adult males. See T. Kohmoto et al., “Effect ofisomaltooligosaccharides on human fecal flora Bifidobacteria,”Microflora, vol. 7, pp. 61 69 (1988). Another study investigated theability of several human gut bacteria to break the α-1,2 and α-1,6glycosidic linkages in α-glucooligosaccharides, in vitro, in substrateutilization tests. See Z. Djouzi et al., “Degradation and fermentationof α-gluco-oligosaccharides by bacterial strains from human colon: invitro and in vivo studies in gnotobiotic rats,” J. Appl. Bact., vol. 79,pp. 117-127 (1995). Branched oligomers were resistant to bothgastrointestinal enzymes and utilization by pathogenic microorganisms.They also reported that α-1,2 glucosidic bonds were more resistant thanα-1,6 linkages in kinetic studies of glucooligosaccharide hydrolysis inpH-regulated fermentations. This study indicated the differences inutilization, and thus effectiveness, of GOS based on types and degree ofbranching.

Production of Glucooligosaccharides

Glucansucrases have been extensively studied because of their role inthe production of dextran and its role in the cariogenic process.Glucansucrases (EC 2.4.5.1), usually extracellular but in some casescell-associated, are primarily produced by various species of soilbacteria. Those produced by Leuconostoc sp. are called dextransucrase.Those produced by Streptococcus sp. and other lactic bacteria,Lactococci, are called glucosyltransferases. Streptococcalglucansucrases synthesize primarily α-1,3 rich polysaccharides.Leuconostoc glucansucrases produce α-1,6 rich polysaccharides.

Glucansucrases catalyze the synthesis of high molecular weight D-glucosepolymers from sucrose. In the presence of efficient acceptors, e.g.,maltose, they may catalyze the synthesis of low molecular weightoligosaccharides. See F. Paul, “Acceptor reaction of a highly purifieddextransucrase with maltose and oligosaccharides: Application to thesynthesis of controlled-molecular-weight dextrans,” Carbohydr. Res.,vol. 149, pp. 433-441 (1986).

Dextransucrases catalyze the synthesis of high molecular weight glucans(dextrans) according to the reaction:

Dextran is a D-glucose polymer composed mainly of α-1,6 linked backbonesin a linear chain and α-1,2, α-1,3, and/or α-1,4 branch linkages. SeeU.S. Pat. No. 5,229,277. The chemical structure of the dextran isspecific to the glucansucrase of the producing strain of microbes (Table1). See J. F. Robyt, “Dextran,” In: Encyclopedia of Polymer Science andEngineering,” (H. F. Mark et al., eds.), Vol. 4, pp. 752-767, John Wiley& Sons, New York (1986). The dextransucrase from L. mesenteroides NRRLB-1299 can produce α-glucooligosaccharides (GOS) containing one or moreD-glucopyranosyl branch units linked via α-1,2 glycosidic bonds ifmaltose supplied as an acceptor. See F. Paul et al., “Method for theproduction of α-1,2 oligodextrans using Leuconostoc mesenteroidesB-1299,” U.S. Pat. No. 5,141,858. However, dextransucrase from L.mesenteroides B-742 (ATCC 13146) produces two dextrans; one with α-1,6and α-1,3 linkages, and another with α-1,6 and α-1,4 linkages. (Table 1)Usually a high molecular weight dextran (10⁶-10⁷ Da) is produced. Thisis the case, for example, of the enzyme from L. mesenteroides NRRL-512F,which is used to produce dextran polymers of industrial interestincluding chromatography supports, photographic emulsions, ironcarriers, and blood plasma substitutes (Robyt, 1986).

TABLE 1 Linkages in different dextrans as obtained by methylationanalysis Linkages % Dextran^(a) Solubility α-1→6 α-1→3 α-1→3 Br^(b)α-1→2 Br^(b) α-1→4 Br^(b) L. m. B-512F Soluble 95 5 L. m. B-742 Soluble50 50 L. m. B-742 Less soluble 87 13 L. m. B-1299 Soluble 65 35 L. m.B-1299 Less soluble 66 1 27 L. m. B-1355 Soluble 54 35 11 L. m. B-1355Less soluble 95 5 S. m. 6715 Soluble 64 36 S. m. 6715 Insoluble 4 94 2^(a)L. m., Leuconostoc mesenteroides; S. m., Streptococcus mutans.^(b)Br, Branch linkage. Adapted from Robyt, 1986.

The synthesis of oligosaccharides using dextransucrase can be induced atthe expense of dextran synthesis. In the presence of sucrose, theintroduction into the reaction medium of molecules, like maltose,isomaltose, and O-α-methylglucoside, shifts the pathway of glucansynthesis towards the production of oligosaccharides. See Paul, 1986;and M. Remaud et al., “Characterization of α-1,3 branchedoligosaccharides synthesized by acceptor reaction with the extracellularglucosyltransferases from L. mesenteriodes NRRL B-742,” J. Carbohyd.Chem., vol. 11, pp. 359-378 (1992); Koepsell et al., 1952; and J. Robytet al., “Relative, quantitative effects of acceptors in the reaction ofLeuconostoc mesenteroides B-512F dextransucrase,” Carbohydr. Res., vol.121, pp. 279-286 (1983). The molecular weight and polydiversity of thisoligosaccharide product are dependent upon the sucrose to acceptorratio, the strain of bacteria, and on the characteristics of theintermediate oligosaccharides in the reaction. The ratio of sucrose tomaltose affects the composition and yield of the oligosaccharidesproduced by the acceptor reaction. When the maltose to sucrose ratio was2, a partially purified dextransucrase from L. mesenteroides NRRL B-512Fproduced 85% of the theoretical yield of polysaccharide asoligosaccharides, with an average degree of polymerization (DP) of 4.See U.S. Pat. No. 5,141,858; and Paul, 1986.

Leuconostoc mesteroides B-742 ATCC 13146

Leuconostoc mesenteroides ATCC 13146 was isolated from spoiledcanned-tomatoes. (Robyt, 1986) The dextran produced by this (B-742)Leuconostoc strain is highly branched, containing as much as 50% α-1,3linkages. Leuconostoc mesenteroides ATCC 13146 actually produces twoexocellular α-D-glucans, a fraction L, which is precipitated at anethanol concentration of 39%, and a fraction S, which is precipitated ata concentration of 45% ethanol (Robyt, 1986). Fraction L consists of anα-1,6 backbone with α-1,4 branch-points, and fraction S consists of anα-1,6 backbone with α-1,3 branch-points. The L fraction from Leuconostocmesenteroides ATCC 13146 contains 87% α-1,6 linkages and 13% α-1,4linkages. The percentage of α-1,3 branch-points in the fraction S glucanis variable, dependant on the conditions under which it is synthesizedfrom sucrose. The α-1,3 linkages of the S fraction of L. mesenteroidesATCC 13146 are all branched linkages. This dextran demonstrates extremeresistance to endodextranase. This property seems related to itsstructure that has the highest possible degree of branching and exhibitsa comb-like structure with main chains of consecutive α-1,6 linkedglucose residues to which single α-1,3 linked glucosyl residues areattached. Any change in reaction conditions that affects the rate ofacceptor reaction relative to chain elongation also affects the degreeof branching in ATCC 13146 fraction S dextran.

The acceptor reaction of L. mesenteroides ATCC 13146 was investigatedand found that branch formation in this strain, when maltose was theacceptor, was dependant upon reaction conditions. L. mesenteroides ATCC13146 in the presence of maltose produced 90% of the theoretical yieldof polymer as isomaltooligosaccharides, under optimum conditions forsucrose fermentation. See Yoo, 1997; S. K. Yoo et al., “Co-production ofdextran and mannitol by Leuconostoc mesenteroides, J. Microbiol.Biotechnol., vol. 11, pp. 880-883 (2001); and S. K. Yoo et al., “Highlybranched glucooligosaccharide and mannitol production by mixed culturefermentation of Leuconostoc mesenteroides and Lipomyces starkeyi, J.Microbiol. Biotechnol., vol. 11, pp. 700-703 (2001). The fermentationwas essentially complete in 24 hours, with oligosaccharide productionbeing linked to growth. The production rate was about 0.9 g/L hr. Themaltose to sucrose ratio was able not only to alter the yield ofoligosaccharide but also to change the relative proportion of differentsize oligosaccharides produced by the fermentation. The highest yieldsof isomaltooligosaccharides were obtained when the ratio of sucrose tomaltose in the fermentation was two. This is the same ratio reported foroptimum oligosaccharide production in vitro by the dextransucrase of L.mesenteroides B-512F (See Paul et al., 1986). Several Leuconostocstrains were tested to check for oligosaccharide size profiles producedin response to maltose, because individual Leuconostoc speciessynthesize different dextransucrases in response to various acceptors.The isomaltooligosaccharides produced by L. mesenteroides ATCC 13146were mostly DP (degree of polymerization) 3-5 by chemical analysis.Isomaltooligosaccharides prepared by alcohol-precipitated, cell-freeculture broths had greater amounts of higher branchedisomaltooligosaccharides up to DP 7, than commercial preparations andhad no glucose and less maltose (Yoo, 1997). Theseisomaltooligosaccharides were found to affect isolated, single microbialcultures by suppressing growth of Salmonella enteritidis, Salmonellatyphimurium, Staphylococcus aureus, Staphylococcus epidermidis, andClostridium perfringenes, and supporting growth of two Bifidobacteriumspecies. (Yoo, 1997).

D-mannitol is a sugar-alcohol derived from mannose or fructose bydehydrogenation. In sucrose fermentations, mannitol is produced as anend product, as fructose can be used as an electron acceptor, but thelevels of mannitol produced vary with the strain. See Yoo, 1997; and C.Y. Kim et al., “Production of mannitol using Leuconostoc mesenteroidesNRRL B-1149,” Biotechnol. Bioprocess Eng., vol. 7, pp. 234-236 (2002).Mannitol was found as one of the major end products in this Leuconostocfermentation. It is necessary to separate the mannitol from theoligosaccharides if they are to be used as prebiotics, because mannitolcan act as an additional carbon source. Its presence would hinder theability to ascribe the essential and unique role of oligosaccharides onintestinal microflora. (Yoo, 1997)

Oligosaccharides as Antibiotic Alternatives in Animals

Antibiotic resistance among known pathogens such as Salmonella andEscherichia coli is expanding due to the wide use of antibiotics inareas ranging from medicine to animal feed. Although only specificantibiotics are used in feed preparations and are exclusive to non-humanuse, their chemical similarity to antibiotics prescribed for humans hasraised concern that resistance will spread more rapidly, since resistantmechanisms generally affect an entire class of antibiotics (ex:penicillinases to inhibit the Penicillins). This, coupled with publicpressure to remove antibiotics from animal feeds, has created a need forsafe alternatives that can effectively control the growth of bacterialpathogens in the human food supply. Selected fructooligosaccharides andglucooligosaccharides have shown potential as alternatives toantibiotics. See P. Monsan et al., (1995); J. V. Loo et al., “Functionalfood properties of non-digestible oligosaccharides: a consensus reportfrom the ENDO project (DGXII AIRII-CT94-1095),” Brit. J. Nutr., vol. 81,pp: 121-132 (1999); and P. Valette et al., “Bioavailability of newsynthesized glucooligosaccharides in the intestinal tract of gnotobioticrats,” J. Sci. Food Agric., vol. 62, pp. 121-127 (1993). However, notall oligosaccharides have been found effective. Although FOS isgenerally considered to be effective in regulating and reducingpathogenic microbial populations, conflicting reports exist about theeffectiveness of GOS. See Naughton et al., 2001; and Yoo, 1997. Theseconflicting reports may be due to variability in the composition of theGOS (the degree of branching, the size, the amount of mannitol, or theacceptor used in fermentation production), or whether the GOS was testedon single microbial cultures, mixed microbial cultures, or in vivo.There may also be differences depending on the animal tested.

During recent years, poultry production and consumption have continuallyincreased. Since 1992, the production of broilers grew from 9,482,000 to14,017,000 tons in 1996 in the United States. Poultry is a carrier ofnumerous bacteria, including Salmonella and Campylobacter. Practicalexperience has demonstrated the difficulty in reducing the incidence ofSalmonella on chickens once they arrive at the processing plant.Significant reduction in Salmonella on processed carcasses requires thedelivery of chickens with reduced Salmonella to the processing plant.One of the possible ways to control Salmonella outbreaks may be throughthe judicious addition of selected carbohydrates to the diet ofchickens. Mannose and lactose in the diet of chickens have been reportedto reduce Salmonella colonization. See B. Oyofo et al., “Effect ofcarbohydrates on Salmonella typhimurium colonization in broilerchickens,” Avian Dis., vol. 33, pp. 531-534 (1989).Fructooligosaccharides (FOS) have been shown to influence intestinalbacterial populations by enhancing the growth of lactic acid bacteriasuch as Lactobacillus species and Bifidobacteria, and to inhibitSalmonella colonization of chicks. See J. S. Bailey et al., “Effect offructooligosaccharide on Salmonella colonization of the chickenintestine,” Poultry Sci., vol. 70, pp. 2433-2438 (1991); and T. Fukataet al., “Inhibitory effects of competitive exclusion andfructooligosaccharide, singly and in combination, on Salmonellacolonization of chicks,” J. Food. Prot., vol. 62, pp. 229-233 (1999).The mean number of Salmonella enteritidis in the chicks of thefructooligosaccharide group was significantly (P<0.05) decreasedcompared with the control group. There are no reports that aglucooligosaccharide is effective in modifying the gut microflora inpoultry.

Importance of α-Glucosidase Inhibition

Starch is one of the most readily available fermentable sources ofenergy for organisms and makes up 60-70% of the dietary carbohydrateconsumption in humans. Humans secrete a pancreatic α-amylase thatcleaves starch to a di-(maltose), tri-(maltotriose), and branchedα-dextrins in the duodenal cavity. Because there is no integraltransport process in the intestinal enterocyte that can accommodateanything larger than free glucose, these oligosaccharides are furtherprocessed to glucose in the intestinal surface membrane by α-glucosylsaccharidases, including α-glucosidase. These enzymes form part of alarge glycoprotein component of the intestinal surface brush bordermembrane. Once formed, glucose then may be cotransported into theenterocyte, along with Nan, either by a 75 kDa specific integral brushborder glucose carrier or by a transporter expressed in the smallintestine. Inhibitors of α-glucosidase are know to delay the digestionof starch, of starch-derived oligosaccharides, and sucrose such that therise in blood sugar levels is slowed and insulin secretion is decreasedafter a meal. These inhibitors have been proposed to be usedtherapeutically for obesity, gastritis, gastric ulcer, duodenal ulcer,caries, hyperglycemia, hyperinsulinemia, diabetes mellitis, cancer,viral infection, hepatitis B and C, HIV and AIDS. See U.S. Pat. Nos.5,840,705; and 4,013,510; and U.S. Patent Application No. 2004/0081711.At least two commercial oral α-glucosidase inhibitors, Miglitol andacarbose, are currently prescribed for use in managingnon-insulin-dependent diabetes mellitus by slowing the appearance ofglucose in the blood after eating.

We have discovered that the isomaltooligosaccharides (IMOs) produced byLeuconostoc mesenteroides ATCC 13146 fermentation with a sucrose tomaltose ratio of 2:1 are effective prebiotics in mixed cultures ofmicrobial populations, including cultures from chicken ceca.Surprisingly in mixed microbial cultures, this IMO composition proved aseffective as FOS as a potential prebiotic. This IMO composition could bean effective alternative to antibiotics for chickens and other poultry.Thus, these IMOs can be used as effective prebiotics for both birds andmammals. Moreover, the IMOs were discovered to be effectivenon-competitive inhibitors of α-glucosidase. These IMOs also will beuseful, as an α-glucosidase inhibitor, in a therapeutic application forseveral diseases, including obesity, diabetes mellitus, pre-diabetes,gastritis, gastric ulcer, duodenal ulcer, caries, cancer, viral diseasesuch as hepatitis B and C, HIV, and AIDS. A diet with 5-20% IMOs wasalso shown to reduce the abdominal fat tissue in mammals.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the structures of various oligosaccharides.

FIG. 2 illustrates the production of various isomaltooligosaccharides byL. mesenteroides ATCC 13146 from sucrose (10% w/v) and maltose (5% w/v)as a function of time.

FIG. 3 illustrates the flow chart for the production ofisomaltooligosaccharides as used in this study.

FIG. 4 illustrates the results of thin layer chromatography indicatingthe types of branched α-isomaltooligosaccharides of L. mesenteroides(ATCC 13146). The abbreviations used are as follows: S,Isomaltodextrins; P, Isomaltooligosaccharide product; C, Commercialisomaltooligosaccharides (Wako Pure Chemical Industry Ltd., Osaka,Japan); Glc, glucose; IM₃, Isomaltotriose; IM₅, Isomaltopentaose; andIM₇, Isomaltoheptaose.

FIG. 5 illustrates the results of ¹³C NMR of the branchedα-isomaltooligosaccharides of L. mesenteroides (ATCC 13146).

FIG. 6 illustrates the anaerobic growth of mixed cultures of Salmonellatyphimurium and Lactobacilli johnsonii on the branchedα-isomaltooligosaccharides of L. mesenteroides (ATCC 13146) preparationat 37° C.

FIG. 7 illustrates the α-glucosidase (maltase) activity inhibition withincreasing concentrations of the branched α-isomaltooligosaccharides ofL. mesenteroides (ATCC 13146).

FIG. 8 illustrates a double reciprocal plot of α-glucosidase (maltase)activity inhibition as the concentration of the branchedα-isomaltooligosaccharides of L. mesenteroides (ATCC 13146) increased.

FIG. 9A illustrates the α-glucosidase (maltase) activity in the presenceof different concentrations of panose (branched; α-1,4 and α-1,6).

FIG. 9B illustrates the α-glucosidase (maltase) activity in the presenceof different concentrations of isomaltotriose (linear; two α-1,6).

FIG. 10A illustrates a double reciprocal plot of α-glucosidase (maltase)activity inhibition as the concentration of panose (branched; α-1,4 andα-1,6) increased.

FIG. 10B illustrates a double reciprocal plot of α-glucosidase (maltase)activity inhibition as the concentration of isomaltotriose (linear; twoα-1,6) increased.

FIG. 11A illustrates the anaerobic growth of Salmonella typhimurium andBifidobacterium longii on different combinations of panose (branched;α-1,4 and α-1,6) at 37° C.

FIG. 11B illustrates the anaerobic growth of Salmonella typhimurium andBifidobacterium longum on different combinations ofmaltooligosaccharides (d.p. 4-10) at 37° C.

FIG. 12 illustrates a comparison of growth rates in log phase betweenSalmonella typhimurium and Bifidobacterium longum at differentconcentrations of panose (branched; α-1,4 and α-1,6) andmaltooligosaccharides (M.O.; d.p. 4-10).

The present invention describes the production and application ofmixtures of isomaltooligosaccharides (IMOs) ranging in size from DP(degree of polymerization) 3 to 7 units and incorporating a maltosylgroup at the reducing end of each oligomer. The said mixtures wereproduced by fermentation with Leuconostoc mesenteroides ATCC 13146 byrestricting the polymer size through the addition of maltose to thecarbon source. A specific ratio of maltose acts to limit the chainlength produced by the enzyme dextransucrase acting on sucrose. The IMOsin this work were produced by a sucrose to maltose ratio of 2:1. Syrupcontaining said fermentation products was obtained after ion exchangeand chromatographic separation of the fermentation broth. Mannitol wasthen removed to produce isolated IMOs. The said mixture produced by thisprocess was found to be readily catabolized by Bifidobacteria andlactobacillus but not readily utilized by either Salmonella sp., or E.coli, pointing towards its use in intestinal microflora modification.The said mixtures were non-competitive inhibitors of α-glucosidase(maltase), an enzyme required for starch or maltodextrin utilization,and decreased the abdominal fat in mammals.

EXAMPLE 1 Materials and Methods

Organism, Culture Medium, and Inoculum Preparation

All strains of bacteria used in this study were obtained from theAmerican Type Culture Collection (ATCC, Manassas, Va.). They weremaintained on agar slants, at 4° C. and transferred monthly. Anaerobeswere subcultured weekly. Salmonella typhimurium (ATCC 14028) andEscherichia coli B (ATCC 23226) were maintained on tryptic soy agar(Difco, Detroit, Mich.). Bifidobacterium bifidum (ATCC 35914),Bifidobacterium longii (ATCC 15708), Lactobacillus johnsonii (ATCC33200), and Leuconostoc mesenteroides (ATCC 13146) were maintainedanaerobically on Lactobaccilli MRS slants (Difco, Detroit, Mich.)containing 0.05% (w/v) cysteine. Chicken ceca were kindly supplied bythe Russell Research Center (USDA ARS, Russell Research Center, Athens,Ga.). Screening and isolation for chicken ceca bacteria were conductedfollowing the method described by R. Hartemink et al., “Comparison ofmedia for the detection of Bifidobacteria, lactobacilli and totalanaerobes from fecal samples,” J. Mircrobiol. Meth., vol. 36, pp.181-192 (1999). Basically, ceca (from 6 weeks to 8 weeks broilers) in aplastic bag were homogenized by kneading the bag, and a subsample ofabout 10 g was transferred to a preweighed glass container containing 90ml anaerobic buffered peptone water (Oxoid) with 0.5 g/L L-cysteine-HCl.The container was then closed and weighed to determine the actual samplesize. Mixed samples were diluted further with reduced physiological saltsolution (“Rps,” peptone 1 g/L, L-cysteine-HCl 0.5 g/L and NaCl 8 g/L)or test media (MRSB; Difco). Finally, the samples were plated on themedia and incubated at 37° C. for 48 h. Unless otherwise stated, mixing,diluting, plating and incubation were carried out anaerobically. Sixcolonies out of the hundreds were selected randomly and designated aschicken ceca isolates #1 to #6.

Preparation of Oligosaccharides

Batch fermentations were conducted in a 2-L BioFlo II fermentor (NewBrunswick Scientific Co.) with a working volume of 1.0 L. The media hadthe following composition: sucrose (100 g/L); maltose (50 g/L); yeastextract (5 g/L); MgSO₄.7H₂O (0.2 g/L); FeSO₄.7H₂O (0.01 g/L); NaCl (0.01g/L); MnSO₄.7H₂O (0.01 g/L); CaCl₂ (0.05 g/L); KH₂PO₄ (3 g/L) with pH7.2. Fermentors were inoculated from late log phase flask cultures at1.0% of working volume. Fermentations were conducted at pH 6.5, 28° C.,and 200 rpm. After harvesting, cells were removed by centrifugation at10,400×g for 20 min (Dupont Sorvall RC5C, Newtown, Conn.). Activatedcharcoal (5 g/L, Sigma Chem. Co., St. Louis, Mo.) and Celite 545 (1 g/L,Fisher Scientific, Fair Lawn, N.J.) were added to cell-free culturebroth and mixed at 50° C. for 20 min. The broths were then filteredthrough No. 6 filter paper (Whatman International Ltd., Maidstone,England) to remove the carbon. The filtered broths were desalted usingion-exchange columns filled with an anion-exchange resin in thehydroxide form and a cation-exchange resin in the hydrogen form (Rohmand Haas, Philadelphia, Pa.). The eluents were concentrated by vacuumevaporation (Brinkmann Instrument Inc., Westbury, N.Y.) to 65% solids.Mannitol crystallized upon cooling the concentrates, and was removed bydecantation. Isomaltooligosaccharides were separated from the mannitolfree concentrates using a cation exchange column (in calcium form); theisomaltooligosaccharide fractions were concentrated by vacuumevaporation.

Analytical Methods

Bacterial growth was measured by turbidimetry at 660 nm, calibratedagainst cell dry weight. Cells from a known volume were harvested bycentrifugation at 10,400×g for 2 min (Dupont Sovall 24S, Newtown,Conn.), washed with deionized water, resuspended in a minimum volume ofwater, and dried (initially overnight at 95° C. and then at 105° C.) toconstant weight. An absorbance of 1.0 at 660 nm was equivalent to 0.51 gof dry matter·liter⁻¹.

Thin Layer Chromatography (TLC)

Separation and qualitative identification of oligosaccharides wasconducted using TLC. Whatman K6F silica gel plates of sizes (10×20 cm)were obtained from Fisher Scientific (Chicago, Ill.). A homologousseries of isomaltodextrins (DP 1-10) was donated by Chonnam NationalUniv. (Kwangju, Korea). Maltopentaose, maltohexaose, maltoheptaose,panose, glucose, and isomaltotriose (Sigma Chem. Co., St. Louis, Mo.)and a commercial mixture of isomaltooligosaccharides (Wako Pure ChemicalIndustry Ltd., Osaka, Japan) were used as standards. Aliquots (1-2 μL)of the solutions to be analyzed were applied 20 mm from the bottom ofthe TLC plates with 10 μL micro syringe pipettes. The plates weredeveloped at ambient temperature, using a mixture of solvents(acetonitrile, ethyl acetate, propanol, and water in volume (ml) atproportions of 85:20:50:70, respectively). After development wascomplete, the plates were dried, and the carbohydrates visualized usinga spray of an ethanol solution containing 0.3% (w/v) α-naphthol and 5%(v/v) H₂SO₄. After air-drying, spots were developed by heating in anoven for 10 to 20 min at 100° C. Isomaltooligosaccharides wereidentified by comparing their chromatographic behavior with those of thestandards.

Cation Column Chromatography

Different types of cation resins (Na, K, Ca form) were tested forseparation of isomaltooligosaccharides from the end fermentationproducts. Resins (Duolite CR-1320, Rohm and Haas, Philadelphia, Pa.) inglass-jacketed columns (10 mm (Inner diameter)×100 mm (Length); workingvolume 70 ml) were regenerated using 5% solutions of NaCl, KCl, or CaCl.The temperature of the water eluent and the circulating water for glassjacket were 92 and 80° C., respectively. No pressure on the column wasapplied. Injection volume was 1 ml of solution (15 Brix° IMO). Thedetector was a differential refractometer (Waters).

High Performance Ion Chromatography

High-performance ion chromatography using a CarboPac MAI column (Dionex,Sunnyvale, Calif.) and a pulsed amperometric detector (PAD, Dionex) wasused for quantitative analysis of glucose, fructose, sucrose, mannitol,and maltose concentrations in solution. The samples were eluted at 0.4ml·min⁻¹ with a 0.48 M NaOH solution. Oligosaccharide concentrationswere calculated from peak areas of high-performance liquidchromatography on an Aminex-HPX-87K Bio-Rad column (Bio-Rad Lab.Hercules, Calif.) run at 85° C. with K₂HPO₄ as eluent, at a constantflow rate of 0.5 ml·min⁻¹, using glucose as a standard.

¹³C Nuclear Magnetic Resonance

The isomaltooligosaccharides (DP 1 to DP 8) were analyzed using a DPX250 (63 MHz ¹³C) system with help of the Department of Chemistry,Louisiana State University (Baton Rouge, La.). The chemical shifts wereexpressed in ppm relative to the methyl signal of acetone in deuteriumoxide solvent which was used as an internal standard at δ=29.92 ppm. Thevarious signals were identified as described by F. Seymour et al.,“Structural analysis of dextrans containing 4-O-α-D-glucosylatedα-D-glucopyranosyl residues at the branch points, by use of 13C-nuclearmagnetic resonance spectroscopy and gas-liquid chromatography-massspectrometry,” Carbohydr. Res., vol. 75, pp. 275 (1979); and M. Remaudet al., “Characterization of α-1,3 branched oligosaccharides synthesizedby acceptor reaction with the extracellular glucosyltransferases from L.mesenteriodes NRRL B-742,” J. Carbohyd. Chem., vol. 11, pp. 359-378(1992).

Kinetic Assay for α-Glucosidase

α-Glucosidase (maltase; EC 3.2.1.20), β-NAD, glucose dehydrogenase (EC1.1.147) and other reagent chemicals were obtained from the SigmaChemical Co. (St. Louis, Mo.). The kinetic assays were based on thefollowing reaction;

The kinetic assays were all performed in 96-well plates and read atwavelength 320 nm in a SPECTRAmax Plus microtiter plate reader(Molecular Devices Corp., Sunnyvale, Calif.) at 37° C. The softwarepackage Softmax™ was used for data analysis. α-Glucosidase (maltase; EC3.2.1.20), β-NAD, and glucose dehydrogenase (EC 1.1.147) solutions wereprepared with 0.1M K₂HPO₄ (pH 7) buffer. Each well contained 25 μl of0.13 IU/ml glucose dehydrogenase, 25 μl of 1.65 IU/ml of α-glucosidase,and 20 μl of 12 mM of G-NAD in a total volume of 200 μl with differentcombinations of sugars and 0.1M K₂HPO₄ (pH 7) buffer. Absorbance changewith time was measured at 320 nm.

Oligosaccharide Utilization by Selected Microorganisms

The growth of selected bacteria in the presence ofisomaltooligosaccharides was compared by measuring absorbance over timeat 660 nm. The media used for both the Bifidobacteria sp. and L.johnsonii was of the same composition as Lactobacillus MRS broth with0.05% (w/v) cysteine, except the carbon source was replaced by variousoligosaccharide preparations. The growth media for S. typhimurium and E.coli was tryptic soy broth, with the carbon source replaced by purifiedisomaltooligosaccharides. Carbon sources were supplied at a finalconcentration of 0.5% (w/v). All carbon sources were filter sterilized(0.2 μm). The following carbon sources were compared: glucose (SigmaChem. Co., St. Louis, Mo.), commercial fructooligosaccharides(FOS; >97.5%, Samyang Genex Co., Seoul, Korea), andisomaltooligosaccharide preparations. Individual culture, anaerobicgrowth tests were conducted in sealed glass test tubes. Each tube wasinoculated from an overnight culture with either S. typhimurium or E.coli and a 24 to 48 hr culture of a Bifidobacteria sp. or L. johnsonii.The experiments with Bifidobacteria sp. and L. johnsonii were conductedunder anaerobic conditions using anaerobic jars (BBL Microbiology Sys.,Cockeysville, Md.) or the Oxyrase plate system (Oxyrase, Inc.,Mansfield, Ohio). MRS broth containing 0.05% (w/v) cysteine witholigosaccharides as a carbon source was used for mixed cultures of S.typhimurium and L. johnsonii. Total viable counts were conducted on MRSagar and the cell numbers of S. typhimurium were determined from growthon MacConkey agar plates (Difco, Detroit, Mich.). The cell numbers forL. johnsonii were obtained as the difference between total viable countand S. typhimurium numbers. TLC was used to determine oligosaccharideconsumption patterns of various strains. The media was MRSB (Difco) forBifidobacteria and ceca bacteria, and TSB (Difco) for S. typhimurium andE. coli containing 0.5% (w/v) of Leuconostoc isomaltooligosaccharidesinstead of glucose as the carbon source. Media pH was adjusted to 6.0and 0.1% (v/v) inoculum grown overnight in MRSB was used. During thegrowth, samples were taken at various times. Samples (2 μl) were appliedon TLC plates.

EXAMPLE 2 Oligosaccharide Production

Isomaltooligosaccharide (IMO) Production by Acceptor Reaction

IMO production by L. mesenteroides ATCC 13146 from sucrose (10% w/v) andmaltose (5% w/v) was followed over time up to about 30 hr. As shown inFIG. 2, IMO production was complete by late log phase, about 10 hrpost-inoculation, and levels did not drop thereafter. Sucrosedisappeared rapidly during log phase of growth, with sucrose depletioncorresponding to the transition point to stationary phase. Once sucrosewas depleted, the accumulated fructose was metabolized to mannitol witha decrease in growth rate compared to growth on sucrose. Fructoseconcentration peaked about the end of log phase then decreased slowly.Mannitol production occurred through the lag phase to the stationaryphase and was linked to the fructose concentration where the rate offructose disappearance was the inverse of the rate of mannitolformation. Oligosaccharide production was associated with cell growth.The conversion of fructose to mannitol was associated with theaccumulation of fructose. Upon completion of fermentation, the cell masswas 3.2 g/L. The weight % yield of oligosaccharide (productproduced×100/[(160×mole of sucrose consumed)+(342×mole of maltoseconsumed)]) was 82% of theoretical, the number 160 in the equation from342 (sucrose M.W.)−((180 (fructose M.W.)+2 (hydrogen M.W.)) and theconversion of fructose to mannitol was 71% of theoretical.

Thin layer chromatography (TLC) clearly showed the course of IMOproduction. (Data not shown). As fermentation proceeded, mono- anddisaccharides disappeared as the higher DP (degree of polymerization)polysaccharides were formed. By 24 hr, all mono- and disaccharides hadbeen converted to higher oligosaccharide polymers. Four mainisomaltooligosaccharides were found. The sizes of these oligomers werecompared with a commercial oligosaccharide product of known composition.The Leuconostoc isomaltooligosaccharides were branched polymers with asize range of DP 2 to 7.

The oligosaccharides, based on their linkages, showed different Rfvalues (Data not shown). The migration of branched isomaltodextrinscontaining single α-1,3 or α-1,4 linkages, was faster than equivalentdextrins containing only α-1,6 linkages, as indicated by J. F. Robyt etal., “Separation and quantitative determination of nanogram quantitiesof maltodextrins and isomaltodextrins by thin-layer chromatography,”Carbohydr. Res., vol. 251, pp. 187-202 (1994). The migration of theLeuconostoc oligosaccharides was faster than equivalent isomaltodextrins(α-1,6 linkages), but slower than equivalent maltodextrins (α-1,4linkages).

Oligosaccharide Separation

The fermentation broth, after cell separation, containedoligosaccharides, mannitol and some organic acids. Because theoligosaccharides are neutral polymers, and the other components (acids,color compounds and salts) are charged, cation resins were used forseparation of oligosaccharides. Neither K⁺ nor H⁺ cation columns clearlyseparated the oligosaccharides from the mannitol and other products,whereas Ca²⁺ cation columns produced two well separated peaks (Data notshown). When analyzed by HPLC as described above, the first peakcontained all the isomaltooligosaccharides, and the second peakcontained mannitol and organic acids, e.g., acetic acid and lactic acid(Data not shown). Based on these results, the process for producing anoligosaccharide product was developed as shown in FIG. 3.

Mannitol Separation

Crystallization at 4° C. separated most of the mannitol fromoligosaccharides as shown by HPLC analysis of the product at differentstages: the broth after deionization, the broth after mannitolcrystallization (86.4% recovered); the mannitol product (>99.0% purity,calculated by the HPLC peak area); and the oligosaccharide product afterthe cation exchange (Ca²⁺ form) column (>98.8% purity). (Data not shown)Pure oligosaccharide solution (14.5 Brix°) was concentrated to 60 Brix°by evaporation. The concentrated pure isomaltooligosaccharides (ca. 60%w/v) were used for the further testing. Table 2 shows the product yieldsat various stages of the production.

Mannitol was a major end product in the Leuconostoc fermentation.However, mannitol must be separated from the oligosaccharides if theyare to be used as prebiotics, as mannitol can also be a carbon sourcefor microorganisms. Most of the mannitol (86.4%) was recovered withoutfurther processing by crystallization at 4° C. To obtain highly purifiedoligosaccharides (>98.8%), a cation exchange column was used. A Ca²⁺resin has high ionic strength and divalent properties, which may accountfor the increased resolution seen when it was used. On a Ca²⁺ resin, theoligosaccharides eluted first followed by a mixture of mannitol andorganic acids (lactic acid and acetic acid). The smaller mannitolmolecule eluted after the oligosaccharides in part because of partialionization of the mannitol at the 6.5 pH. Divalent cations such as Ca²⁺bind strongly to the organic acids. At pH 6.5, which is above the pKvalue of lactic and acetic acids, they exist in dissociated forms. Thestronger organic acid that is lactic (pK of 3.79) eluted later becauseit interacts more strongly with Ca²⁺ than acetic acid, pK value of 4.7.

TABLE 2 Product yields and process for production ofglucooligosaccharides Process Components in process Fermentation InputSucrose  100 g/L (10% w/v) Maltose (5% w/v)   50 g/L Yeast extract 8.28g/L and salts

Output Oligosaccharides 80.1 g/L (82.3%^(a)) Mannitol 37.6 g/L(70.1%^(b)) Acids, ethanol 5.75 g/L and cell mass Decolorization anddeionization

Removal of color pigments and salts Evaporation Concentrated to ca. 60%(w/v) Crystallization at 4° C. Oligosaccharides, Mannitol (86.4%residual mannitol recovered, 99.0% purity) Ca ²⁺Ion exchange

Purified oligosaccharides 98.8% purity ^(a)Weight % yield ofoligosaccharide (product produced × 100/[(160 × mole of sucroseconsumed) + (342 × mole of maltose consumed)]) ^(b)% fructose conversionto mannitol

EXAMPLE 3 Composition and Structure of Isomaltooligosaccharide Products

Thin layer chromatography (as described in Example 1) showed that IMOwere branched polymers ranging in size from DP 2 to 7 (FIG. 4). By HPLCpeak area, there was 6.9% DP 2, 28.4% panose, 36.7% branched DP 4, 19.1%branched DP5, 7.4% branched DP6, and 1.2% branched DP7. In the pureform, there was only a trace amount of monosaccharides (<0.2%) present,and no polysaccharides larger than DP 7.

Structural analysis of IMOs by C¹³ NMR (as described in Example 1)showed that the IMOs are linked mainly by α-1,4 and α-1,6 linkages (FIG.5). These oligosaccharides were analyzed using a DPX 250 (63 MHz ¹³C)system. The chemical shifts in FIG. 5 are expressed in ppm relative tothe methyl signal of acetone in a deuterium oxide solvent, which wasused as an internal standard at δ=29.92 ppm. The various signals wereassigned as described by Seymour et al. (1976) and Remaud et al. (1992):85-105 ppm, the anomeric region (mainly 97-103 ppm, as there is only aninfinitesimal proportion of reducing sugar in any of the polymers);70-75 ppm, C-2,3,4 and 5; 60-70 ppm, bonded and non-bonded C-6 atoms;75-85 ppm, signals of bonded C-2, C-3, C-4, C-5.

Two closely separated peaks at 100.44 ppm were also encountered in thespectrum of maltose, and both correspond to a glucose molecule linked toa reducing residue of maltose by an α-1,4 linkage (See Remaud et al.,1992). This also implied that the α-1,4 linkage is located at thereducing end of isomaltosyl residues containing α-1,6 linkages. Thepeaks corresponding to the region of 98.0-99.0 ppm showed α-1,6 linkedresidues. However, the intensity of the resonances for α-1,3 bondsaround 100.0 and 80.6-81.2 ppm were not present (Dols et al., 1998;Remaud et al., 1992).

The isomaltooligosaccharides produced were branched polymers between DP2 and 7 in size. Prior researchers had reported that theoligosaccharides synthesized by the dextransucrase from this bacteriumhad α-1,6 backbones with α-1,3, and/or α-1,4-branched side chains whenmaltose was used as an acceptor. See Remaud et al. (1992). However,under the current conditions, the IMOs produced contained mainly α-1,4and α-1,6 linkages and maltose was linked to the reducing end of theisomaltosyl residues.

EXAMPLE 4 Isomaltooligosaccharides as Microbial Growth Modifiers

Individual Cultures

Growth of selected bacteria on L. mesenteroides ATCC 13146isomaltooligosaccharides as a carbon source was compared with growth ona commercial fructooligosaccharide (FOS) mixture. Both types ofoligosaccharides produced significantly reduced growth of S. typhimuriumand E. coli compared with growth on glucose (Table 3). Based on the TLCanalysis of the medium at different times, these organisms could not usethe IMOs efficiently (Data not shown). There was no significantdifference between growth rates on either of the oligosaccharidepreparations. The growth rate suppression of E. coli in the presence ofIMOs was marginally greater than that of S. typhimurium (Table 3). Thegrowth of selected probiotic strains on IMOs was also compared.Leuconostoc IMO supported the growth of Bifidobacterium longum and L.johnsonii and showed no significant difference when compared to glucoseas carbon source. B. longum degraded almost all components of the IMOwithin 24 hrs as shown by TLC (Data not shown). Utilization of the IMOproduct by B. bifidium was less rapid (74.9% relative to growth rate ofFOS) than utilization of a commercial FOS and glucose. This indicatesthat the growth of probiotic strains was also dependent on the type ofoligosaccharides.

TABLE 3 Growth comparison on isomaltooligosaccharide preparations: IMO,Leuconostoc isomaltooligosaccharides; FOS, Commercialfructooligosaccharides (Samyang Genex Co., Seoul, Korea) Growth rate inexponential growth phase Growth rate ([Absorbance unit × 100] · hr⁻¹)(on IMO/ Organism glucose IMO FOS glucose) S. typhimurium 9.89 3.64 3.4836.8 E. coli 9.35 2.68 2.44 28.7 B. bifidium 13.30 9.81 13.10 73.8 L.johnsonii 11.06 10.74 10.70 97.1 B. longum 11.72 11.69 11.70 99.7

Mixed Cultures

To test for prebiotic effects of the IMO, mixed cultures of S.typhimurium and L. johnsonii were grown on the oligosaccharides. FIG. 6shows the anaerobic growth of the two mixed cultures over 30 hr, as afunction of time and medium pH. When the medium pH was above 5.0, bothorganisms grew; however, S. typhimurium grew more slowly than L.johnsonii. As the population of L. johnsonii increased, the pH dropped.When the pH dropped below 5.0, S. typhimurium populations decreaseduntil they were below detection level (<1).

When S. typhimurium or E. coli was grown on ATCC 13146 IMO preparations,there was less than 37% of the equivalent growth on glucose, similar togrowth on commercial fructooligosaccharides (less than 35%). The factthat these bacteria showed similar growth on IMO and on FOS issurprising based on the literature. Lactobacillus johnsonii and B.longum showed no differences in growth rate on glucose or the IMOpreparations. When L. johnsonii and S. typhimurium were grown togetheron oligosaccharide preparations, the oligomers stimulated the growth ofthe Lactobacillus, but were not readily utilized by the Salmonella. TLCshowed clearly that the IMOs preferentially stimulated the growth ofBifidobacterium, but were not readily utilized by Salmonella and E.coli. It appears that these IMOs are selectively favored by someprobiotic strains.

EXAMPLE 5 Utilization of IMOs by Bacteria Isolated from Chicken Ceca

Utilization of Leuconostoc IMOs by six bacterial isolates (all showedGram positive, catalase negative and lactic acid formation from glucose)from chicken ceca was compared to utilization of a commercialfructooligosaccharide (FOS). Three of the six bacteria showed moregrowth after 24 hr on IMOs than on FOS (Table 4). In mixed cultures ofbacteria from chicken ceca, the cecal bacterial isolates #5 and #6showed the same use pattern of IMOs. Only the DP 3 polymer (panose) wasutilized in the first 24 hours as shown by TLC (Data not shown).

TABLE 4 Growth comparison of chicken cecal bacteria on varioussubstrates: glucose, IMO, Leuconostoc isomaltooligosaccharide; and FOS,Commercial fructooligosaccharides (Samyang Genex Co., Seoul, Korea)Relative growth to glucose as a carbon source at 24 hr incubation([Absorbance unit of Glc at 24 hr/Absorbance unit of × 100] · hr⁻¹)Organism glucose IMO FOS C.B. # 1 100.00 22.77 61.20 C.B. # 2 100.0099.34 79.29 C.B. # 3 100.00 75.85 48.01 C.B. # 4 100.00 36.72 61.40 C.B.# 5 100.00 87.23 50.25 C.B. # 6 100.00 87.17 87.66 ^(a)Growth level atstationary phase (at 24 hr) on glucose was calculated as 100. C.B.;Cecal Bacterium

To test the potential of this IMO as a prebiotic in poultry, sixdifferent microbial strains were isolated from chicken ceca. Theseisolates were identified as lactic acid bacteria by colonial morphologyand chemical reaction (Gram positive, catalase negative, and lactic acidformation from glucose, data not shown). When utilization of theLeuconostoc isomaltooligosaccharides by these isolates was compared withutilization of a commercially available fructooligosaccharide (FOS),surprisingly three of the six isolates showed better growth after 24 hron IMO than on FOS. In tests of mixed cultures of these lactic acidbacteria and Salmonella on the IMOs, five of the six cecal isolatesshowed higher growth rates and inhibited the growth of Salmonella.Similar results were seen with Lactobacillus and Bifidobacteriumstrains. Two isolates showed identical patterns of consumption of IMO.They only degraded the DP 3 component of the IMO mixture. Similareffects have been seen in studies on the effect offructooligosaccharides in feed trials with broilers where FOS reducedsusceptibility of poultry to Salmonella colonization, increasedBifidobacterium levels, and reduced the level of Salmonella present inthe caecum. See Bailey et al. (1991); and Chamber et al. (1997).

The low pH produced by the chicken cecal bacteria is likely responsiblefor the observed suppression of S. typhimurium growth in mixed cultures.Although other antagonistic substances such as bacteroicins and hydrogenperoxide could be produced that can inhibit S. typhimurium, significantlevels of lactic acid bacteria must be generated first. These studiesdid not directly measure in vivo effects of IMO as produced in thisstudy, but indicated that this IMO composition can be effective as anavian prebiotic.

EXAMPLE 6 Inhibition of α-Glucosidase by IMOs

α-Glucosidase and IMO

The Leuconostoc IMO was found to inhibit the activity of α-glucosidase(maltase). FIG. 7 shows the inhibition of α-glucosidase activity withincreasing concentrations of IMO (0%, 0.25%, and 0.5%) as measured atvarious concentrations of maltose (0, 50, 100, 250 mM). A doublereciprocal plot of this data indicated this was a non-competitiveinhibition (FIG. 8).

In order to determine the role of branching in the inhibition, the Kivalues for panose and isomaltotriose were also determined. FIGS. 9A and9B shows the inhibition of α-glucosidase activity with increasingconcentration of panose and isomaltotriose, respectively (0%, 0.25%, and0.5%), as measured at various concentrations of maltose (0, 50, 100, 250mM). A double reciprocal plot of this data is shown in FIGS. 10A and10B. Only panose, containing α-1,4 and α-1,6 linkages, showed aninhibition on α-glucosidase. Isomaltotriose, a linear glucose polymerlinked by two α-(1→6) linkages, was not inhibitory.

Growth Test of B. longum and S. typhimurium with Panose andMaltodextrins

To further determine whether Leuconostoc IMO acts as a starch-metabolisminhibitor, B. longum and S. typhimurium were grown in differentcombinations of panose and maltooligosaccharides (from DP 4 to DP 10).As the concentration of panose in the growth medium increased, thegrowth of S. typhimurium slowed but B. longum growth increased. (FIGS.11A and 11B, respectively). A comparison of growth rates at log phaseclearly showed the growth inhibition of S. typhimurium by panose (FIG.12). In the case of B. longum, 50% panose+50% maltooligosaccharides and75% panose+25% maltooligosaccharides combinations showed better growththan other combinations.

L. mesenteroides ATCC 13146 IMOs were found be a non-competitiveinhibitor of α-glucosidase (maltase). To verify inhibition ofα-glycosidase by branched oligomers, panose and isomaltotriose weretested for inhibition. Panose contains α-1,4 and α-1,6 linkages and isone of the components in the Leuconostoc IMOs. Panose inhibitedα-glucosidase, whereas isomaltotriose, containing two α-1,6 linkages ina linear structure, did not. Panose also suppressed growth of S.typhimurium but not B. longum. B. longum showed increased growth whenpanose and maltodextrins were supplied in the medium together comparedwith maltodextrin alone. When growth rates at early log phase werecompared, growth inhibition of S. typhimurium by panose was clearlyevident.

Isomaltooligosaccharides (branched or partially branched) most likelyinhibit some of those enzymes required for utilization of starch inother genera, such as Escherichia and Salmonella. Panose and theLeuconostoc isomaltooligosaccharides reduced the activity ofα-glucosidase that degrades α-1,4 linkages in a maltose or maltodextrin.Panose alone did not produce a higher growth rate thanmaltooligosaccharides and panose together for B. longum. It is likelythat high concentrations of panose also can inhibit enzymes seen inmaltooligosaccharide inhibition. There seems to be a synergistic effectfor the probiotic strains on carbon source utilization when maltodextrinand prebiotic sugars are present together.

EXAMPLE 7 Isomaltooligosaccharides as Dietary Supplement for ChicksInoculated with Salmonella

Young chickens were used to test the effectiveness of these branchedisomaltooligosaccharides (IMO) produced as described in Example 2 as adietary supplement to reduce Salmonella intestinal infections. Youngchickens (commercial Leghorn broiler chickens on day of hatch) wereorally inoculated with Salmonella using a round tip cannula attached toa syringe containing nalidixic acid-resistant Salmonella. This uniquestrain was used to be able to distinguish these bacteria from thegeneral population. The chickens were then divided into four groups tobe fed standard chicken feed (prepared and milled by the poultrydepartment of the University of Georgia) with added concentrations ofIMOs of 0, 1, 2 and 4% (w/w). On day 21, the chickens were sacrificed toexamine the ceca (large intestine) and count the bacterial populationsof Salmonella, Bifidobacteria, Lactobacillus, and total anaerobicbacteria. In addition, the weight gain efficiency was determined, andthe general condition of the birds noted. There was no significantdifference in weight gain efficiency between chickens fed IMO in thefeed and control. There was a 0.1 pH unit drop in cecal pH in the birdsreceiving IMO at all concentrations.

Based on the above data (Examples 4 and 5) and the drop in pH, it ispredicted that the IMO-supplemented food will be effective as aprebiotic, i.e., will increase the numbers of beneficial bacteria(Bifidobacteria and Lactobacillus) and decrease the numbers ofpathogenic bacteria (Salmonella). Thus IMO-supplemented food would beuseful as an antibiotic for poultry.

EXAMPLE 8 Toxicity Study of Isomaltooligosaccharides

To test whether the isomaltooligosaccharide composition produced asdescribed in Example 2 is toxic to mammals, young rats were used andvarious body organs assayed after several weeks of feedingIMO-supplemented food. Young male Sprague-Dawley rats (about 2 monthsold, mean weight of 270 g) were used. The rats were divided into fourgroups of 5 to 6 rats per group. One group (the control) was fedstandard rat chow (Purina rat chow). The other three groups were fedIMO-supplemented rat chow at a concentration of 5%, 10%, and 20%,respectively. The food intake and weight gain was measured twice a weekfor six weeks. At the end of six weeks, the rats were sacrificed toexamine the weights of the major organs.

There were no significant differences in food intake (although a trendtoward an increase in the IMO food intake was seen; p<0.058). Weightgain, heart weight, spleen weight, kidney weight, lung weight, brownadipose tissue weight, and white adipose tissue weight were determined.(Data not shown) There were significant differences in the weight of thecaecum with an increased weight measured especially in the 10% and 20%IMO groups. This probably indicates an increase in the population offermentation bacteria. Blood was also taken for future analysis.

There was also a significant effect of the IMO concentration on theabdominal fat gain when normalized for food intake. A significantdecrease was seen in abdominal fat with increasing levels of IMO in thefeed. (Table 5)

TABLE 5 Accumulation of abdominal fat in rats after 6 weeks at variousconcentration of isomaltooligosaccharides Concentration ofIsomaltooligosaccharides Abdominal fat (gm)/ (gm IMO/gm food) FoodIntake (gm) 0 0.0012 0.05 0.00075 0.10 0.00063 0.20 0.000316

These data indicate that IMO-supplemented food is non-toxic. Moreimportantly, this indicates that IMO-supplemented food can reduce eitherthe formation or deposition of fat. It is also predicted that the bloodglucose level will be less in rats fed the IMO-supplemented food.

EXAMPLE 9 Effect of Isomaltooligosaccharides-Supplemented Food on BloodGlucose

To determine the effectiveness of IMOs produced as described in Example2 on blood sugar levels after eating, rats will be used. The rats willbe fed IMO-supplemented food as described above in Example 8, and theblood glucose levels monitored overtime after feeding. It is expectedthat the blood sugar levels in the IMO-fed rats will rise at a slowerrate than the controls based on the data above showing that these IMOsare effective α-glucosidase inhibitors (Example 6). It is also predictedthat the insulin level will be decreased. This indicates that these IMOswould be effective therapeutically for diabetes or pre-diabetes.

The complete disclosures of all references cited in this application arehereby incorporated by reference. Also, incorporated by reference is thecomplete disclosure of the following documents: Chang-Ho Chung, “Apotential Nutriceutical from Leuconostoc mesenteroides B-742 (ATCC13146); Production and Properties,” A dissertation submitted to theDepartment of Food Science, Louisiana State University, May 2002; and D.F. Day and Chang-Ho Chung, “Probiotics from Sucrose,” a slidepresentation at the May 22, 2002 meeting of the American Society ofMicrobiologists. In the event of an otherwise irreconcilable conflict,however, the present specification shall control.

1. A composition for decreasing the population numbers of pathogenicbacteria in a bird's intestine, said composition comprising a commercialavian food and two or more isomaltooligosaccharides, wherein eachisomaltooligosaccharide has at least one α-1,4 linkage and at least oneα-1,6 linkages, has a degree of polymerization between 2 and 7, and hasmaltose at the reducing end; wherein said isomaltooligosaccharides areproduced by fermentation of sucrose in the presence of maltose, in asucrose:maltose ratio of about 2:1, by Leuconostoc mesenteroides ATCC13146; and wherein the isomaltooligosaccharides are at a concentrationfrom about 0.1% to about 20% w/w food.
 2. A composition as in claim 1,wherein the isomaltooligosaaccharide concentration is from about 0.1% toabout 4% w/w food.
 3. A method to decrease the populations numbers ofpathogenic bacteria in a bird's intestine, comprising orallyadministering to the bird the composition as in claim
 1. 4. A method asin claim 3, wherein the bird is a chicken.
 5. A method as in claim 3,wherein the pathogenic bacteria is one or more selected from the groupconsisting of Salmonella, Escherichia, and Campylobacter.
 6. A method asin claim 5, wherein the pathogenic bacteria is Salmonella.